Ionizing radiation is a well-known human carcinogen linked to a variety of cancers including thyroid cancer, leukemia, breast cancer and soft tissue sarcomas. The molecular mechanisms of radiation-induced carcinogenesis remain poorly understood. Significant evidence has been accumulated supporting the dominant role of chromosomal rearrangements in the carcinogenesis initiated by radiation exposure. Since ionizing radiation induces double strand DNA breaks, it is conceivable that chromosomal rearrangements are formed directly by mis-rejoining of free DNA ends produced by radiation and located close to each other in the nucleus. In thyroid cancer, RET/PTC rearrangements, which usually form via an inversion in chromosome 10q, are a molecular signature of radiation-associated tumors. Recently, we have established an in vitro model of dose-dependent generation of RET/PTC in human thyroid cells after exposure to radiation. This system will serve as an important tool for further studies of radiation-induced DNA damage and chromosomal rearrangements. In this proposal, we will test the hypotheses that RET/PTC rearrangement is a direct result of mis-rejoining of double-strand DNA breaks induced by radiation exposure. Specifically, we will characterize the frequency and spectrum of radiogenic breaks and rejoining kinetics in the RET gene region, test if one or two DNA breaks are required for the rearrangement, and compare the effects of irradiation and 1-131 on the generation of RET/PTC rearrangements. We will also determine the fate of cells after they acquire the rearrangement and will characterize the earliest stages of radiation-induced carcinogenesis initiated by various RET/PTC types. Finally, we will find whether these rearrangements are predisposed by altered function of several genes playing a central role in double-strand break repair in vitro. Then, we will test the role of alterations in these genes in predisposition to radiation-induced thyroid cancer in human populations. These studies will expand our understanding of the mechanisms of radiation-induced carcinogenesis and provide important information for radiation risk assessment and protection applicable to a variety of areas such as clinical use of external radiotherapy and I-131, occupational radiation exposure, space exploration, and radioprotection from potential accidents involving nuclear power reactors or radiological terrorism.